1. Field of the Invention
The present invention features methods and compositions for identifying compounds which inhibit ApoCI and which are useful in the treatment or prevention of atherosclerosis, plaque rupture, apoptosis, or myocardial infarction. The invention further features methods for treating subjects suffering from or at risk of developing atherosclerosis, plaque rupture, apoptosis, or myocardial infarction. The invention further features methods for diagnosing subjects at suffering from or at risk of developing treatment or prevention of atherosclerosis, plaque rupture, apoptosis, or myocardial infarction.
2. Background
Over the last decade, considerable body of evidence indicates that premature plaque rupture due to apoptotic death of aortic smooth muscle cells (ASMC) in the fibrous cap is a major contributor to the pathological sequelae of atherosclerosis, i.e. arterial thrombosis (Godfrey K M and Barker D J P (2000) Am. J. Clin. Nutr. 71(Suppl):1344S-352S; Kwiterovich, Jr., P O. et al. (2002) Am. J. Card. 90(Suppl 8A):1i-10i; Kwiterovich Jr P O. et al. (In press, 2004) Ethn. Dis.) leading to myocardial infarctions (Diaz M. et al. (1989) Metabolism. 38:435-8) or stroke (Kaser S. et al. (2001) Metabolism. 50:723-8). Other studies based upon the use of carotid artery biopsies and immunohistochemical techniques reveal that macrophages and T lymphocytes are the major cell type found closely associated with the sites of plaque rupture in human subjects (Diaz M. et al. (1989) Metabolism. 38:435-8). Caspases are cystein-aspartate specific proteases and contribute critically in the final phase of apoptosis i.e. executing the cleavage of DNA, an irreversible process in apoptosis. Both caspase-1 and caspase-3 have been implicated to contribute to the execution phase of apoptosis in vivo in human atherosclerotic plaques (Merzouk H. et al. (1997) Acta. Paediatr. 86:528-32; Radunovic N. et al. (2000) J. Clin. Endocrin. Metab. 85:85-88).
Apolipoprotein C-I (ApoCI), a 6.6-kDa single-chain plasma protein of 57 amino acids, has a basic pI because of its high content of lysine (16 mol %) and contains no histidine, tyrosine, cysteine, or carbohydrate (Jong, M. C. et al. (1999) Arterioscler. Thromb. Vasc. Biol. 19:472-484; Shachter, N. S. (2001) Curr. Opin. Lipidol. 12:297-304). Residues 7 to 24 and 35 to 53 of ApoCI are important for binding to plasma lipids (Jong, M. C. et al. (1999) Arterioscler. Thromb. Vasc. Biol. 19:472-484; Shachter, N. S. (2001) Curr. Opin. Lipidol. 12:297-304). ApoCI is a component of very-low-density (VLDL), intermediate density, and high-density lipoproteins (HDL). ApoCI displaces apolipoprotein E (apoE) from VLDL and intermediate density, thereby decreasing their clearance from plasma (Windler, E. E. and Havel R. J. (1985) J. Lipid Res. 26:556-565). ApoCI decreases the binding of β-VLDL to a remnant receptor, the low-density lipoprotein (LDL) receptor-related protein (LRP) (Kowal R. C. et al. (1990) J. Biol. Chem. 265:10771-10779; Weisgraber, K. H. et al. (1990) J. Biol. Chem. 265:22453-22459), and apoE-mediated binding of VLDL and intermediate density to the LDL receptor (LDLR) (Windler, E. E. et al. (1980) J. Biol. Chem. 255:10464-10471; Sehayek, E. and Eisenberg, S. (1991) J. Biol. Chem. 266:18259-18267). ApoCI inhibits cholesterol ester transfer protein (Gautier, T. et al. (2000) J. Biol. Chem. 275:37504-37509) and phospholipase A2 activity (Poensgen, J. (1990) Biochim. Biophys. Acta 1042:188-192). ApoCI stimulates lecithin cholesterol acyl transferase to ˜80% of that of apolipoprotein A-I (apoA-I) (Soutar, A. K. et al. (1975) Biochemistry 14:3057-3064).
Human ApoCI-transgenic mice, with a wild-type background or with a knockout background for the LDLR or apoE, manifest a marked combined hyperlipidemia because of significantly delayed remnant clearance (Shachter, N. S. et al. (1996) J. Clin. Invest. 98:846-855; Jong, M. C. et al. (1998) J. Clin. Invest. 101:145-152; Jong, M. C. et al. (2001) Diabetes 50:2779-2785; Jong, M. C. et al. (1996) J. Clin. Invest. 98:2259-2267; Jong, M. C. et al. (1999) Biochem. J. 338:281-287; Jong, M. C. et al. (1996) Arterioscler. Thromb. Vasc. Biol. 16:934-940; Conde-Knape, K. et al. (2002) J. Lipid Res. 43:2136-2145). Free fatty acid levels are elevated because of reduced fatty acid uptake in peripheral tissues, which is an effect that is paradoxically associated with increased sensitivity to insulin and protection from obesity (Jong, M. C. et al. (1998) J. Clin. Invest. 101:145-152; Jong, M. C. et al. (2001) Diabetes 50:2779-2785). Of particular interest here, Conde-Knape et al (Conde-Knape, K. et al. (2002) J. Lipid Res. 43:2136-2145), using a moderately expressing ApoCI transgenic on apoE-null background to study the effect of ApoCI independent of apoE, found a marked combined dyslipidemia that included an ApoCI-enriched HDL and increased atherosclerosis. ApoCI-enriched HDL (but not VLDL) had a marked inhibitory effect on hepatic lipase (Conde-Knape, K et al. (2002) J. Lipid Res. 43:2136-2145). ApoCI knockouts are normolipidemic rather than hypolipidemic (van Ree, J. H. et al. (1995) Biochem. J. 305:905-911). Cholesterol ester transfer protein-transgenic/apoCI knockout mice manifest a markedly increased transfer of cholesteryl esters from HDL to VLDL (Gautier, T. et al. (2002) J. Biol. Chem. 277:31354-31363).
In humans, Bjorkegren et al reported a significant enrichment of ApoCI in VLDL remnants in normolipidemic patients with coronary artery disease and exaggerated postprandial triglyceridemia (Bjorkegren, J. et al. (2000) Circulation 101:227-230) and in healthy, normolipidemic men with early asymptomatic atherosclerosis (Bjorkegren, J. et al. (2002) Arterioscler Thromb Vasc Biol. 22:1470-1474).